Our work focuses on the mechanisms by which transcription factors regulate the development of diverse hematopoietic lineages, and how these factors orchestrate terminal differentiation with cell cycle arrest. These functions require key nuclear factors to be able to both activate and repress transcription. GATA-1 activates as many genes as it represses during terminal erythroid differentiation in a manner largely dependent on the interaction with its co-factor FOG-1. We recently identified two novel mechanisms by which GATA-1 and FOG-1 regulate transcription. First, we purified a FOG-1-associated co-represser complex NuRD (nucleosome remodeling and deacetylase) that is required for GATA-1/FOG-1-mediated transcriptional repression. Second, using chromosome conformation capture (3C) we showed that GATA-1 and FOG-1 are required for the formation of long-range chromatin loops at the p-globin locus. In Aim I we propose to study the function of the FOG-1-NuRD complex at the molecular and cellular level as well as in murine models. This includes the identification of the modules that mediate NuRD binding by FOG-1, determining their atomic structure, testing the function of NuRD subunits in erythroid cell lines, generating mice with mutations that disrupt the FOG-1-NuRD interaction, and the conditional knock out of the NuRD core subunit Mi-2p\ Since FOG-1 can function as co-activator and co-repressor for GATA-1, Aim II examines the mechanisms by which FOG-1 switches between these opposing functions. These studies include the analysis by CHIP and siRNA of NuRD components at activated and repressed genes. This Aim will also investigate the role of posttranslational modifications of FOG-1 and NuRD with regard to the switch in activity. Based on encouraging preliminary results, in Aim III we will examine by 3C whether GATA-1 and FOG-1 actively form chromatin loops during the repression of the GATA-2 and c-kit genes. This requires a thorough prior characterization of the GATA-2 and c-kit loci with regard to GATA-1 binding and histone acetylation. The role of loop formation will be directly addressed in functional studies. We hope that together these studies will help to explain how key transcriptional regulators can switch between activating and repressive functions. This knowledge directly impacts on how nuclear proteins promote the formation of one lineage at the expense of another, and how they activate genes associated with the mature phenotype while simultaneously inhibiting a program that maintains the immature proliferative state.